Protocols - SOPs

DNA Melting Curve

1. SYBR Green I preparation

a. Dilute 1�L of stock SYBR (10,000x) into 10mL 1xTE to make 1x SYBR Green

2. DNA preparation

a. Add 100ng DNA to SYBR buffer

b. Total volume of 30�L per tube

c. Whatever volume you do not need for DNA or any salt you want to add should be taken up by SYBR, with at minimum around 10�L SYBR

3. Load samples into microplate tray, being careful not to contaminate tray

4. Cover tray with “Microseal ‘B’ Film”: a transparent plastic adhesive film

5. Write melting curve program on thermal cycler protocol editor

a. Hold at 50C for 5 minutes

b. Add “Melting curve” step, and adjust variables: 50� C to 98� C, 0.2� increments, 20 seconds each step, with plate read

c. Add another “Melting curve” step, going in the opposite direction – 98� C to 50� C

6. Add plate details

a. In the “Plate” tab, make a program with the lanes your samples are in selected

b. Make sure SYBR Green is selected as the fluorophore

7. Remove metal tray in thermal cycler and insert the plastic tray with your samples, making sure it is oriented according to the lanes you selected in 6a

8. Run the program – sit back and wait!